Project Summary Coproheme decarboxylase (ChdC, also known as HemQ) catalyzes the final step in a recently delineated pathway for heme b biosynthesis in critically important Gram-positive pathogens like Staphylococcus aureus, Mycobacterium tuberculosis, and Bacillus anthracis. ChdC binds and activates two molecules of H2O2 at its substrate coproheme iron center, leading to the sequential oxidative decarboxylation of the propionate side chains at porphyrin ring positions 2 and 4 (P2 and P4) to form heme b. Previous studies have inferred a ChdC mechanism in which the oxidation is mediated by a nearby tyrosine. This tyrosine is first oxidized by an ill- defined high-valent heme species in what is thought to be a proton-coupled electron transfer reaction to yield the corresponding tyrosyl radical (Y?). Evidence suggests that H-atom transfer from the ring-adjacent methylene carbon of P2 or P4 to Y? sets up the last oxidation step by the heme FeIV=O to release CO2 and form the 2- or 4- vinyl group. The aims of the project are threefold: 1) Understand the mechanism by which coproheme III is transferred from ferrochelatase (CpfC) to ChdC; 2) Elucidate the nature and mechanistic roles of the prompt and heretofore unidentified reactive coproheme- and harderoheme-centered intermediates in the ChdC-catalyzed reaction of coproheme III with H2O2; and 3) Examine the mechanistic steps unique to the oxidative decarboxylation of the harderoheme III P4 group. The first aim will identify and assess the protein- protein interactions required to transfer the coproheme from CpfC to ChdC without suffering the toxic effects of releasing it into the cell. This aim will be addressed using reactivity assays, kinetic studies, variants of ChdC and CpfC, and biophysical methods, including analytical ultracentrifugation and native gradient gel electrophoresis. The second two aims will be addressed with spectroscopic (resonance Raman, electron paramagnetic resonance, and transient absorbance), kinetic (stopped flow and freeze-quench), and cyroradiolytic sample preparation methods to gain insight into the structural and electronic properties of the ChdC prompt intermediates that direct their reactivities toward product. S. aureus is a leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis. Paramount to our national health is the timely development of effective clinical strategies against the growing threat of drug-resistant bacterial infections. Thus, ChdC and CpfC from S. aureus will be developed as a paradigm for understanding the final step in heme b biosynthesis and the role of protein protein complexation in protecting the cell from off-pathway reactivity and toxicity of hemes in Gram-positive pathogens. Since the ChdC proteins are only found in Gram- positive bacteria and not in humans and as heme is essential during infection, ChdC holds promise as a yet unexploited target for antimicrobial therapeutics, once the mechanistic aspects of its function are understood.